Blocking the angiopoietin-2–dependent integrin β-1 signaling axis abrogates small cell lung cancer invasion and metastasis

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin β-1–dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin β-1 or blocking Integrin β-1 signaling by an anti–ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin β-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1–targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin β-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.


Title
Blocking the Angiopoietin-2-dependent Integrin beta-1 signaling axis abrogates small cell lung cancer invasion and metastasis.S2.SCLC-bearing mice were treated with vehicle control (black; n=10), IgG control (grey; n=7), VEGFR inhibitor monotherapy (VEGFRi, dark blue; n=6), anti-Angiopoietin-2 monotherapy (aANG-2, light blue; n=6), anti-PD-1 monotherapy (aPD-1, orange; n=10), and anti-Angiopoietin-2/VEGFR inhibitor/anti-PD-1 triple combination therapy (triple, pink; n=10).(A) Log of the hazard ratio of the different therapy cohorts to show the suitability of the proportional hazard model for overall survival analysis.The received therapy is indicated by the color code.(B) Log of the hazard ratio of different therapy cohorts to show the suitability of the proportional hazard model for progression-free survival analysis.The received therapy is indicated by the color code.(C) Synergy analysis regarding overall survival of the triple combination with anti-Angiopoietin-2, VEGFR inhibitor and anti-PD-1 using the proportional hazard model and the R Survival package.P-values ≤ 0.05 are indicated as significant.nsnot significant.(D) Synergy analysis regarding progression-free survival of the triple combination with anti-Angiopoietin-2, VEGFR inhibitor and anti-PD-1 using the proportional hazard model and the R Survival package.P-values ≤ 0.05 are indicated as significant.nsnot significant.(E) Randomization of therapy groups in regards to target lesion diameter at the start of therapy, target lesion diameter at the end of therapy and the age at death.The received therapy is indicated by the color code.Statistical analysis was done using the Student's t-test (ns, not significant; *, p < 0.05; **, p < 0,01; ***, p <0,001; error bars, SEM).(F) Survival probability of the overall survival displaying the observed (blue) and expected (yellow) survival using the proportional hazard ratio model.(G) Survival probability of the progression-free survival displaying the observed (blue) and expected (yellow) survival using the proportional hazard ratio model.P-values ≤ 0.05 are indicated as significant.nsnot significant.(vehicle; n=9), IgG control (IgG; n=7), VEGFR inhibitor monotherapy (VEGFRi; n=6), anti-ANG-2 monotherapy (aANG-2; n=6), anti-ANG-2 and VEGFR inhibitor combination therapy (aANG-2/VEGFRi; n=5), anti-PD-1 monotherapy (aPD-1; n=6), and anti-ANG-2/VEGFR inhibitor/anti-PD-1 triple combination therapy (triple; n=8).

Figure S2 .
Figure S2.ITGB1 expression significantly correlates with migration markers in human SCLC cell lines.

Figure S3 .
Figure S3.ITGB1 protein expression of human SCLC cell lines.

Figure S4 .
Figure S4.Vimentin expression in liver metastasis of SCLC patients.

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Figure S5.TIE-2 receptor expression is not altered upon extensive stage of disease on SCLC cells in vivo.

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Figure S6.ANG-2 is not significantly increased in SCLC liver metastasis.

Figure S7 .
Figure S7.ITGB1 signaling is needed for extravasation of SCLC cells.

Figure S9 .
Figure S9.ITGB1 expression correlates with SCLC migration capacity and ANG-2 and Fibronectin provide similar transcripts associated to cell migration.

Figure S11 .
Figure S11.ADAM9 is responsible for SCLC cell migration and regulated downstream of ITGB1.

Figure S15 .
Figure S15.Murine primary SCLC tumors mimic the expression of NCAM and KI-67.

Figure S16 .
Figure S16.Murine primary SCLC tumors show increased T cell infiltration.

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Figure S17.Murine SCLC liver metastases mimic the expression of NCAM and KI-67.

Figure
Figure S18.Murine SCLC liver metastases show increased T cell infiltration.

Figure
Figure S1.ITGB3, ITGA5 and ITGA1 positively correlate with tumor stage in SCLC.Human SCLC RNA-Seq data is analyzed regarding integrin correlations with tumor stage.Association between integrin gene expressions and UICC tumor stage is tested using the nonparametric Jonckheere-Terpstra test (n=75).

Figure S2 .
Figure S2.ITGB1 expression significantly correlates with migration markers in human SCLC cell lines.(A)mRNA expression data for SCLC provide the "migration" and "apoptosis" gene sets in human SCLC cell lines (n=64).The heatmap is generated by the heatmap tool provided by SCLC-CellMiner.The correlation to ITGB1 expression is shown.(B) Pearson correlation coefficients with ITGB1 expression in human SCLC cell lines of all genes per "migration" and "apoptosis" gene set (p-values from one-sample Wilcoxon-tests for median corr.coefficient equal to zero per gene set; *, p < 0.05; **, p < 0,01; ***, p <0,001).Data of mRNA gene expression and statistical analysis is listed in in the Supplemental TableS2.

Figure S3 .
Figure S3.ITGB1 protein expression of human SCLC cell lines.Surface expression of ITGB1 was analyzed in selected human SCLC cell lines from different tissues of origin by flow cytometry.Histogram indicates one representative experiment out of three.Error bars indicate SEM.

Figure S4 .
Figure S4.Vimentin expression in liver metastasis of SCLC patients.FFPE lung tumor material of six SCLC patients and matched liver metastasis was analyzed for the expression of Vimentin.Bars indicate 60 µm.

Figure S5 .
Figure S5.TIE-2 receptor expression is not altered upon extensive stage of disease on SCLC cells in vivo.(A-C) Relative expression of the integrins CD49e (A) and CD61 (B) and TIE-2 (C) on SCLC tumor cells isolated from lung (n=6-9) and liver (n=6), normalized to IgG control, determined by flow cytometry.Origin of tumor cells, presence of metastasis and stage are indicated as limited stage of disease (LD) and extensive stage of disease (ED).Statistical analysis was done using the Student's t-test (ns, not significant; *, p < 0.05; **, p < 0,01;***, p < 0,001; error bars, SEM).(D) Human SCLC RNA Seq data (n=81) was analyzed regarding TEK (encoding for TIE-2) correlations.(E) Association between TEK expression and UICC tumor stage were tested using the non-parametric Jonckheere-Terpstra test (n=75).

Figure S7 .
Figure S7.ITGB1 signaling is needed for extravasation of SCLC cells.Representative images of H&E and NCAM IHC stain (n=5) of SCLC WT and ITGB1-KO orthotopically injected into the lung.Bars indicate 100 µm.(B) Scheme of i.v.injection of SCLC cells to determine extravasation capacity.Created with BioRender.com.(C) The capability of wildtype SCLC clones and ITGB1-KO to form tumors after i.v.injection was determined by IHC based on H&E and NCAM and quantified for lungs and livers, respectively (n=5).

Figure S9 .
Figure S9.ITGB1 expression correlates with SCLC migration capacity and ANG-2 and Fibronectin provide similar transcripts associated to cell migration.(A) SCLC liver metastasis cells (Scheme created with BioRender.com)were stimulated with Angiopoietin-2 "A" and Fibronectin "F" and compared to control "O" by RNASeq regarding migration genes.(B) ADAM9 counts per million (CPM) of normalized RNA Sequencing data of ITGB1 knock-outs (n=3) vs controls (n=3) are shown.(C) Cell counting assay for 72h of SCLC cells obtained from liver metastasis and ITGB1KO generated by CRISPR Cas9.One representative experiment out of three.(D) ITGB1 protein expression was determined by flow cytometry on generated murine SCLC cell lines 5 passages after generation.SCLC cell migration upon Fibronection stimulation for 24 h determined by scratch assay.Images after 0 h and 24 h were analyzed using ImageJ.(E) Cell counting after 72h stimulation with fibronectin or ANG-2 of SCLC cells obtained from liver metastasis and ITGB1KO generated by CRISPR Cas9.One representative experiment out of three.Statistical analysis was done using the Student's t-test (ns, not significant; *, p < 0.05; **, p < 0,01; ***, p < 0,001; error bars, SEM).

Figure S11 .
Figure S11.ADAM9 is responsible for SCLC cell migration and regulated downstream of ITGB1.(A) ADAM9 is knocked down using two different siRNA.(B+C) The area of migrated SCLC tumor cells isolated from wild type liver metastasis with ADAM9 (WT) and without ADAM9 (ADAM9-KD) was determined by scratch assay.Cells were stimulated with Angiopoietin-2 (grey) for 24h and compared to control (ctrl).Representative images out of three experiments.Images after 0h and 24h were analyzed using ImageJ.(D) ADAM9 RNA expression data of SCLC patients (n=81) was correlated with ITGB1 gene expression using univariate analysis.Pearson correlation coefficient (r) and p-value are indicated.(E) ADAM9 RNA expression data of for human SCLC cell lines (n=64) in the SCLC CCLE-Broad-MIT data set provided by

Figure S13 .
Figure S13.Targeting Angiopoietin-2-dependent ITGB1 signaling counteracts a VEGF/VEGFR-induced metastatic SCLC phenotype.(A+B) Different models and conditions of VEGF/VEGFR signaling inhibition were analyzed for liver metastases.The average number of microscopic liver metastasis per 2mm 2 was determined by scanned H&E slides in an autochthonous mouse model of SCLC and in a

Figure S15 .
Figure S15.Murine primary SCLC tumors mimic the expression of NCAM and KI-67.SCLC-bearing mice were treated and upon detection of progressive disease based on mouse adapted RECIST v1.1 criteria determined by µCT, endpoint analysis was performed using IHC.NCAM and KI-67 IHC stains on FFPE SCLC tissue of primary lung tumors.Images were taken at 40x magnification.Bars indicate 50 μm.Representative images for each condition are shown:

Figure S16 .
Figure S16.Murine primary SCLC tumors show increased T cell infiltration.SCLCbearing mice were treated and upon detection of progressive disease based on mouse adapted RECIST v1.1 criteria determined by µCT, endpoint analysis was performed using IHC.CD31, CD4 and CD8 IHC stains on FFPE SCLC tissue of primary lung tumors.Images were taken at 40x magnification.Bars indicate 50 μm.Arrows indicate T cells of interest.Representative images for each condition are shown: (vehicle; n=9), IgG control (IgG; n=7), VEGFR inhibitor

Figure S17 .
Figure S17.Murine SCLC liver metastases mimic the expression of NCAM and KI-67.SCLC-bearing mice were treated and upon detection of progressive disease based on mouse adapted RECIST v1.1 criteria determined by µCT, endpoint analysis was performed using IHC.NCAM and KI-67 IHC stains on FFPE SCLC tissue of SCLC liver metastases.Images were taken at 40x magnification.Bars indicate 50 μm.Representative images for each condition are shown: vehicle (n=7), IgG control (IgG; n=6), VEGFR inhibitor monotherapy (VEGFRi; n=6),

Figure S18 .
Figure S18.Murine SCLC liver metastases show increased T cell infiltration.SCLC-bearing mice were treated and upon detection of progressive disease based on mouse adapted RECIST v1.1 criteria determined by µCT, endpoint analysis was performed using IHC.CD31, CD4 and CD8 IHC stains on FFPE SCLC tissue of SCLC liver metastases.Images were taken at 40x magnification.Bars indicate 50 μm.Arrows indicate T cells of interest.Representative images for each condition are shown: vehicle (n=7), IgG control (IgG; n=6),